Molecular biotechnology is a discipline that is based on the ability of researchers to transfer specific units of genetic information from one organism to another. This process, known as cloning, relies on the techniques of recombinant DNA technology to produce a useful product or a commercial process (Glick, B. R.; Pasternak, J. J., Molecular Biotechnology Principles and Applications of Recombinant DNA, 2nd ed. American Society for Microbiology, Washington, D.C. 1998).
Commercial processes often require that proteins encoded by the cloned genes are produced at high rates of expression. There is no single strategy for achieving maximal expression of every cloned gene. Most cloned genes have distinctive molecular properties that require the investment of considerable time and effort before a specific set of conditions that result in an appropriate level of expression is found. There are a variety of ways to modulate gene expression. Microbial metabolic engineering generally involves the use of multi-copy vectors to express a gene of interest under the control of a strong or conditional promoter. Increasing the copy number of cloned genes generally increases amounts and activity of encoded enzymes, therefore allowing increased levels of product formation that is important to commercial processes. However, it is sometimes difficult to maintain vectors in host cells due to instability. Deleterious effects on cell viability and growth can be observed due to the vector burden. The introduction and expression of foreign DNA in a host organism often changes the metabolism of the organism in ways that may impair normal cellular functioning. This phenomenon is due to a metabolic load or burden imposed upon the host by the foreign DNA. The metabolic load may result from a variety of conditions including: 1) increasing plasmid copy number, 2) overproduction of proteins, 3) saturation of export sites, and/or 4) interference of cellular function by the foreign protein itself. It is also difficult to control the optimal expression level of desired genes on a vector. Several reports have suggested altering the copy number of plasmids can have benefit in production of recombinant protein and analysis of transcriptional fusions (Grabherr et al., Biotech. Bioeng., 77:142-147 (2002); Podkovyrov, S. M. and Larson, T. J., Gene, 156:151-152 (1995)).
Bacterial plasmids are extrachromosomal genomes that replicate autonomously and in a controlled manner. Many plasmids are self-transmissible or mobilizable by other replicons, thus having the ability to colonize new bacterial species. In nature, plasmids may provide the host with valuable functions, such as drug resistance(s) or metabolic pathways useful under certain environmental conditions, although they are likely to constitute a slight metabolic burden to the host. To co-exist stably with their hosts and minimize the metabolic load, plasmids must control their replication, so that the copy number of a given plasmid is usually fixed within a given host and under defined cell growth conditions.
The number of copies of a plasmid can vary from 1, as in the case of the F plasmid, to over a hundred for pUC18. Bacterial plasmids maintain their number of copies by negative regulatory systems that adjust the rate of replication per plasmid copy in response to fluctuations in the copy number. Three general classes of regulatory mechanisms have been studied in depth, namely those that involve directly repeated sequences (iterons), those that use only antisense RNAs (AS-RNA), and those that use a mechanism involving an antisense RNA in combination with a protein.
Several chromosomal genes are known to affect the copy number of certain groups of plasmids. The pcnB gene encoding the poly(A) polymerase I has been found to affect copy number of ColE1 plasmids in Escherichia coli. Mutations in the pcnB locus of E. coli reduce the copy number of ColE1-like plasmids, which include pBR322-derived plasmids (Lopilato et al., Mol. Gen. Genet., 205:285-290 (1986)) and pACYC-derived plasmids (Liu et al., J. Bacteriol., 171:1254-1261 (1989)). Furthermore, it was discovered that the pcnB gene product was required for copy number maintenance of ColE1 and R1 plasmids of the IncFII compatibility group. Copy number of R1 plasmids like ColE1 is controlled by an antisense RNA mechanism, though the mechanism is different between the two. The iteron-regulated plasmids F and P1 were maintained normally in strains deleted for pcnB.
The gene relA encoding (p)ppGpp synthetase 1 allows cells to initiate stringent response during starvation. ColE1-type of plasmids can be amplified in amino acid-starved relA mutants of Escherichia coli (Wrobel et al., Microbiol Res., 152:251-255 (1997)). Differential amplification efficiency of plasmids pBR328 (pMB1-derived replicon) and pACYC184 (p15A-derived replicon) was observed in the relA mutant during starvation for particular amino acids.
A recent paper described an origin-specific reduction of ColE1 plasmid copy number due to specific mutations in a distinct region of rpoC (Ederth et al., Mol. Gen. Genomics, 267:587-592 (2002)). The specific mutations, including a single amino acid substitution (G1161R) or a 41-amino acid deletion (Δ1149-1190), are located near the 3′-terminal region in the rpoC gene, encoding the largest subunit β′ of the RNA polymerase. These mutations cause over 20- and 10-fold reductions, respectively, in is the copy number of ColE1. The RNA I/RNA II ratio, which controls the ColE1 plasmid copy number, was affected by these mutations.
The problem to be solved is to identify and provide chromosomal gene modifications that alter plasmid copy number in bacteria. The present invention has solved the stated problem through the discovery that disruptions in any one of 5 (thrS, rpsA, rpoC, yjeR, and rhoL) chromosomal genes will result in increase of copy number of certain plasmids. The effect of mutation of these loci on plasmids is novel and could not have been predicted from known studies.